#*''The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.''

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#*''It may be that the thaw process needs to take place at 37&deg;C rather than in slushy ice.''<cite>PierceCellLysis</cite>

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#*''An alternative option is to use the bead beater to mechanically lyse the cells.''

*Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields.

*Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields.

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*Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column.

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*Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column. However, consider using a SlyD knockout strain. SlyD is a 20-25 kDa protein that has several histidines near each other and can often contaminate Ni column purifications from ''Escherichia coli''.

*20 year old spin columns don't work. :)

*20 year old spin columns don't work. :)

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*The Sauer lab tends to use a higher salt concentration in the lysis, wash and elution buffers. It may give a better wash and may be useful with DNA binding proteins.

The Qiagen protocols calls for you to sonicate or homogenize on ice to lyse cells six times for 10s each time with 5s pauses in between. However sonication is difficult in small volumes and tends to heat up your sample. Freeze thaw cycles should be sufficient to lyse the cells.

It may be that the thaw process needs to take place at 37°C rather than in slushy ice.[1]

An alternative option is to use the bead beater to mechanically lyse the cells.

Notes

Using the Qiagen Ni-NTA resin may be preferable for proteins with low yields.

Contaminating proteins tend to be less of an issue in bacteria because there are few proteins with neighboring histidines that tend to bind to the column. However, consider using a SlyD knockout strain. SlyD is a 20-25 kDa protein that has several histidines near each other and can often contaminate Ni column purifications from Escherichia coli.

20 year old spin columns don't work. :)

The Sauer lab tends to use a higher salt concentration in the lysis, wash and elution buffers. It may give a better wash and may be useful with DNA binding proteins.

Can lyse cells by doing repeated freeze-thaw cycles at -80°C or sonication also works.